The ubiquitin system is a major regulatory mechanism of cellular processes in which speed, specificity, and timing are critical. Ubiquitin-mediated proteolysis of key substrates controls cell cycle progression, signal transduction pathways, differentiation, apoptosis, DNA repair and the immune response. This process is mediated by a multimeric machine, composed of a regulatory ubiquitin-targeting component and an effector protein degradation engine. The regulatory component, which targets ubiquitin to proteins destined for degradation, is itself composed of several multimeric elements (e.g., the SCF ubiquitin ligase complexes) that contribute much of the specificity inherent in the process. In humans, there are sixty-eight SCF ligases, each characterized by a different F-box protein subunit that provides specificity by directly recruiting the substrate to the rest of the ligase and, ultimately, to the ubiquitin-conjugating enzyme. Notably, only three out of 68 human SCF ubiquitin ligases (containing the F-box proteins UTrcp, Fbw7 and Skp2, respectively) have well-established substrates, many of which are involved in cell cycle control. The remaining 65 F-box proteins are considered as "orphan" since their substrates still await discovery. We have recently developed a novel immunopurification strategy that enriches for substrates of F-box proteins followed by mass spectrometry analysis. We will systematically identify biologically significant substrates of human orphan F-box proteins (Specific Aim 1). Because of our research interest, we will focus particularly on those orphan F-box proteins that our preliminary results suggest to be involved in cell cycle control and cancer. Under Aim 2, we will validate the biologically most significant substrates identified under Aim 1. Given their critical role in regulating cell proliferation, SCF ligases are often the target of cancer- related deregulation and involved in oncogenic transformation. Therefore, the information gained from the proposed studies will be of direct relevance to cancer biology and other proliferative diseases. [unreadable] [unreadable] [unreadable]